Compounds and their compositions having anti-inflammatory and anti-thrombotic activities

ABSTRACT

Compounds and their compositions, of general formula: A--X 1  --NO 2  are used as medicaments wherein: A=R(COX) t  t=0 or 1; X=O and the remaining substituents are defined in the specification.

This application is a 371 of PCT/EP96/04696 filed Oct. 29, 1996.

The present invention relates to new products having anti-inflammatory,analgesic and anti-thrombotic activities.

In particular it relates to inhibitors of cyclo-oxygenase (COX) of theclass of Aspirin, i.e. of acetylsalicidic acid or its derivatives ingeneral.

It is known that the anti-inflammatory and anti-thrombotic efficacy ofNSAIDs (Non Steroid Anti-Inflammatory Drugs), also known as FANS, butabove all their tolerance, seem to be markedly affected by theirinhibitor activity of the cyclo-oxygenase (COX) in the inflammatory siteas well as in healthy tissue. See for example FASEB Journal 1, 89, 1987;Bioch. Biophys. Acta 1083, 1, 1991. It is generally believed that thestronger a COX inhibitor is the more effective it is.

The disadvantage of these products is that they are toxic.

Furthermore, it is also known that the COX-inhibiting properties seem todepend on some factors bound to the physico-chemical and structuralcharacteristics of the molecules themselves, such as for example theacidic function. See for example J. Pharmacol. Exp. Therap. 196, 226,1976; Arch. Toxicol. 60, 261, 1987.

The known cyclo-oxygenase inhibitors are generally acids which can bebrought back to general structures, including:

carboxyl acids, either acetylated such as, for example, aspirin andtriflusal, or nonacetylated such as, for example, salycilate,diflunisal, salsalate;

acetic acids, for example diclofenac, indomethacin, tolmetin, sulindac,etodolac, ketorolac;

propionic acids, such as, for instance, ibuprofen, naproxen, pirprofen,tiaprofenic acid, loxoprofen, indoprofen, oxaprozin, ketoprofen,fenoprofen, fenbufen, flurbiprofen, carprofen, suprofen.

See for example a previous patent application in the name of theapplicant PCT/EP 95/01233, herein incorportated by reference, whichdescribes the prior art of the above products.

As said, the disadvantage of these products is that they are veryeffective but highly toxic.

The importance of the acidic function resides in the fact that themasking of this function in COX inhibitors results in a virtuallycomplete loss of its prostanoid-inhibiting properties. See Drugs 35,504, 1988.

Products are also known which are highly effective in inhibitingcyclooxygenase and have a low toxicity even though they do not containthe acidic function in their molecule.

These products are known as nitric esters with nonacidic ending. See forexample patents WO 94/04484, which describes a particular group ofcompounds including the well known commercial product diclofenac; WO94/12463, which describes another specific group of compounds includingthe commercial products flurbiprofen and indoprofen, PCT/EP 94/03182,which describes another specific group of compounds including thecommercial products naproxen and ketorolac.

In a previous patent application in the name of the applicant PCT/EP95/01233 other nitric esters having a nonacidic termination have beendescribed with various linking groups X₁ as specified below. The newlinking groups therein described showed advantages from thepharamcological and pharmaceutical viewpoint, in particularpharmaco-cinetic and pharmaco-dynamic viewpoint, since they showed alower variability of the response. The products described in said patentapplication were also able to exert an inhibition effect of theinflammation produced by lyposaccaride (LPS) and therefore useful in theseptic shock. This result was unexpected since it is well known that theanti-inflammatory products in general do not significantly modify theactivity of the nitrosynthetase induced by lypopolysaccarides in the ratand therefore they are not useful in the septic shock.

The technical problem to be solved by the present invention relates toinhibitor products of the COX much more effective in inhibiting thepiastrine aggregation induced by arachidonic acid and trombin, thelatter having a well known primary patogenetic role even superior toarachidonic acid and other aggregant stimulus, said products havingcontemporaneously a high gastric tolerability, without provokingadhesions of the gastric intestinal mucose on the treated animals.

The applicant has unexpectedly and surprisingly found a specific classof anti-inflammatory products, as described hereinbelow, having animproved inhibtor activity of the COX combined with a low toxicity.

An object of the present invention are compounds, or their compositions,of general formula:

    A--X.sub.1 --NO.sub.2

or their salts, for use as medicaments, in particular asanti-inflammatory and antithrombotic agents, having improved efficiencyin inhibiting the piastrinic aggregation induced by arachidonic acidand/or trombin, wherein:

A=R(COX)_(t), wherein t is zero or 1;

X=O, NH, NR_(1C) wherein R_(1C) is a linear or branched alkyl having 1to 10 C atoms, preferably 1-4 C atoms, or ##STR1## wherein m and n areintegers from 1 to 6, preferably m from 1 to 3, and n from 2 to 4;R_(2a) being H, CH₃ ; the linking with X₁ can be in any position of thering, preferably in position 2; --OCOR₃ preferably in position orthowith respect to --COX₀ --;

X₀ =X;

R is chosen from: ##STR2## wherein: R₁ is an OCOR₃ group, wherein R₃ ismethyl, ethyl or a linear or branched C₃ -C₅ alkyl, or the residue of aheterocycle with a single ring having 5 or 6 atoms which may bearomatic, partially or totally hydrogenated, containing one or moreheteroatoms independently chosen from O, N, and S;

R₂ is hydrogen, hydroxy, halogen, a linear or when permissible branchedalkyl having 1 to 4 C atoms, a linear or when permissible branchedalkoxyl having 1 to 4 C atoms, a linear or when permissible branchedperfluoroalkyl having 1 to 4 C atoms, for example trifluoromethyl;nitro, amino, mono- or di-alkylamine in which the alkylamine has 1 to 4C atoms;

R₁ and R₂ together are a dioxymethylene group, with the proviso thatwhen X=NH, then X₁ is ethylene and R₂ =H; R₁ cannot be OCOR₃ in position2 when R₃ is methyl; nI being 0 or 1;

X₁ in the formula A--X₁ --NO₂ is a bivalent connecting bridge chosenfrom the following:

    --YO--

where Y is selected from:

a linear or when permissible branched C₁ -C₂₀ alkylene, preferablyhaving from 1 to 3 carbon atoms;

a cycloalkylene having from 5 to 7 carbon atoms optionally substituted;##STR3## wherein n is an integer from 1 to 6, preferably from 2 to 4;R_(2a) as defined above; r=0 or 1; Y as defined above, preferably C₁-C₁₀, preferably C₂ -C₆.

The preferred products according to the present invention are those inwhich t=0, X₀ is oxygen; the group having NO₂ is in position 2 withrespect to --COX₀ ; nI=0; R₃ =CH₃. In particular the preferred productsaccording to the present invention are the following:

R₁ is an OCOR₃ group, wherein R₃ is methyl, etnyl or a linear orbranched C₃ -C₅ alkyl, or the residue of a heterocycle with a singlering having 5 or 6 atoms which may be aromatic, partially or totallyhydrogenated, containing one or more heteroatoms independently chosenfrom O, N, and S;

R₂ is hydrogen, hydroxy, halogen, a linear or when permissible branchedalkyl having 1 to 4 C atoms, a linear or when permissible branchedalkoxyl having 1 to 4 C atoms, a linear or when permissible branchedperfluoroalkyl having 1 to 4 C. atoms, for example trifluoromethyl;nitro, amino, mono- or di-alkylamine in which the alkylamine has 1 to 4C atoms;

R₁ and R₂ together are a dioxymethylene group, with the proviso thatwhen X=NH, then Y is ethylene and R₂ =H; R₁ cannot be OCOR₃ in position2 when R₃ is methyl; nI being 0 or 1

X₁ in the formula A--X₁ --NO₂ is a bivalent connecting bridge chosenfrom the following: ##STR4##

where Y is selected from:

a linear or when permissible branched C₁ -C₂₀ alkylene, preferablyhaving from 1 to 3 carbon atoms;

a cycloalkylene having from 5 to 7 carbon atoms optionally substituted;ned in X). All well known synthetic routes for forming these bonds maybe used to form this connection.

In the case of the esters, the most direct synthetic route involves areaction of acyl chlorides R--CO--Cl with halogen alcohols for exampleHO--Y--Cl, HO--Y--Br, HO--Y--I, in the experimental conditions wellknown in the art.

The reaction products are converted into the final products by reactingwith AgNO₃ in acetonitrile, in accordance to what known from theliterature.

The general route is as follows:

    R--CO--Cl+HO--Y--Br→R--CO--O--Y--Br+AgNO.sub.3 →A--X.sub.1 --NO.sub.2

wherein X₁ =YO.

In the case of amides the synthetic route involves a reaction of saidacyl chlorides RCOCl with amino alcohols of the general formula NH₂--Y--OH, NHR_(1C) --Y--OH to give amides of the general formula:

    R--CO--NH--Y--OH and R--CO--NHR.sub.1C --Y--OH

in accordance with known methods.

The reaction of said amides with halogenating agents such as, forexample, PCl₅, PBr₃, SOCl₂, etc., brings to halogen derivatives of thegeneral formula:

    R--CO--NH--Y--Br(Cl) and R--CO--NR.sub.1C --Y--Br(Cl).

The latter products by reacting with AgNO₃ in acetonitrile in accordancewith known literature methods, bring to the final products A--X₁ --NO₂.

The route may be outlined as follows: ##STR5## wherein YO is X₁.

An alternative route to form the esters is a reaction of the sodium orpotassium salts of the acids with the nitric esters of halogen alcoholsof the general formula:

    NO.sub.2 --O--Y--Cl (Br, I)

to directly give the products of the invention.

The reaction route is as follows:

    R--CO--ONa+Br--Y--ONO.sub.2 →R--CO--O--Y--ONO.sub.2

wherein YO is X₁.

The following examples are being given only as illustrative explanationbut not as a limitation of the present invention.

EXAMPLES Example 1

Comparison--Preparation of the Products

It was used acetylsalicilic acid ASA available on the market, Aspirin ofBayer.

Example 2

Comparison--Preparation of the Compound A--X₁ --NO₂, wherein R has theformula below of Aspirin, X₁ is --(CH₂)₄ O--, herein called ANBE, andhaving general formula:

2-acetoxy-benzoate of (4-nitroxy)butyl ##STR6## Preparation of theintermediate having formula: 2-acetoxy-benzoate of (4-bromine)butyl##STR7## At a solution of:

    ______________________________________                                        acetylsalicilic acid  15.0 g and                                              dimethylformamide       50 ml                                                 ______________________________________                                    

kept at 0° C. under nitrogen stream it is added portionwise: 2.6 g ofNaI (80% by weight suspension in vaseline oil).

The mixture was left under stirring for 1 hour and then was dropped in 5hours, at 25° C. in a stirred solution of:

    ______________________________________                                        2,2'-dibromo-butane   27.0 g and                                              dimethylformamide       50 ml                                                 ______________________________________                                    

The mixture was left under stirring for 3 days, then was dried atreduced pressure. The residue was treated with:

    ______________________________________                                        water                  50 ml                                                  dichloromethane        50 ml                                                  ______________________________________                                    

The phases were separated and the aqueous phase was further extracted in10 ml of dichloromethane.

The pooled organic phases were washed with water (3×25 ml), dried(MgSO₄), decoloured with animal charcoal (1 g), and brought to drynessin vacuum.

The residue (26.0 g) was used crude for the next reaction.

Preparation of ANBE

At a solution of

    ______________________________________                                               ASA--(CH.sub.2).sub.4 Br                                                                      26.0 g                                                        acetonitrile      65 ml                                                ______________________________________                                    

kept at room temperature and sheltered from light, was added

    ______________________________________                                               silver nitrate 21.0 g                                                  ______________________________________                                    

After 2 days under stirring were added 4.3 g of silver nitrate.

After 2 further days under the same conditions the insoluble salts werefiltered and the filtrate was freed of the solvent at reduced pressure.

A residue of 18.0 g was obtained and the chromatography on a silica gelcolumn (500 g of silica) eluting with a toluol/ethyl acetate 95/5 v/vmixture was carried out.

The fractions resulted uniform for TLC (Thin Layer Chromatography)analysis and were pooled and brought to dryness and gave 15.0 g of ANBE.

The ¹ H NMR (CDCl₃) (80 MHz) analysis showed the following data: 2.28(3H, s); 1.2 (4H, m); 4.30 (2H, t); 4.50 (2H, t); 7.3 (3H, m); 7.95 (1H,dd). The IR analysis (Nujol) provided the following results: υ_(OCO)=1780 cm⁻¹ ; υ_(COO) =1725 cm⁻¹ ; υ_(ONO2) =1641 e 1287 cm₋₁. Massspectrometry gave a molecular weight value of 297.

Example 3

Preparation of the compound A--X₁ --NO₂ wherein R has the formula below,X₁ is --(CH₂)O, herein called ANMPE having formula:

2-acetoxy-benzoate of (3-nitroxymetyhyl)fenyl ##STR8## Preparation ofthe intermediate of formula ##STR9## In a 1 l flask were added:

    ______________________________________                                        3-OH-benzyl alcohol  28.1 g (0.226 mols)                                      Methylene chloride     85 ml                                                  HBr (48% by weight in water)                                                                        140 ml                                                  ______________________________________                                    

and were kept under stirring at room temperature for 1 hour and half.

At the end the phases were separated and the aqueous phase was furtherextracted with methylene chloride (about 50 ml).

The pooled organic phases were washed twice with:

    ______________________________________                                        Distilled water          100 ml                                               Solution of NaHCO.sub.3 at 5% (w/v)                                                                     50 ml                                               ______________________________________                                    

Then it was anhydrified on MgSO₄ and was brought to dryness obtaining aresidue equal to 34.13 g of crystalline solid.

The product was characterized by TLC analysis, by using atoluol/ethylacetate 7/3 v/v mixture as eluent.

The so obtained product is used immediately for the following reaction.

In a 1 l flack provided with stirrer, termometer, dropping system wereadded:

    ______________________________________                                        Previous reaction residue                                                                              34 g                                                 Acetonitrile            100 ml                                                ______________________________________                                    

In the dropping system it was charged a solution of:

    ______________________________________                                               Silver nitrate 38.5 g                                                         Acetonitrile     60 ml                                                 ______________________________________                                    

and it was dropped in about 2 hours, keeping the flask sheltered fromlight and cooling on a water bath.

The temperature was maintained between 20 and 30° C.

It was left to react for about 15 hours.

Then it was filtered and the filtrate was dried; at the residue wasadded etylacetate, about 500 ml, then silica (50 g) and coal (3 g).

The filtrate was dried again and a charomatography on about 300 g ofsilica using toluol as eluent was carried out by using thechromatographic system indicated above.

11.7 g of product were obtained (dark oil) and characterized by TLC.

Preparation of ANMPE ##STR10##

In a 250 ml flask provided with stirrer, termometer, dropping systemwere introduced:

    ______________________________________                                        3-hydroxybenzylnitrate 4.95 g                                                 potassium carbonate     7.0 g                                                 etylacetate              50 ml                                                ______________________________________                                    

It was cooled at 0° C. and dropped under nitrogen stream in 15 minutes asolution of:

    ______________________________________                                        acetylsaliciloil chloride                                                                            5.01 g                                                 ethylacetate             20 ml                                                ______________________________________                                    

At the end of the dropping it was left to react for about 4 hours at 20°C.

The reaction was made in TLC (toluol ethylacetate 9/1 v/v).

At the end 70 ml of distilled water was added.

The phases were separated, the aqeuous phase was extracted again with 30ml of ethylacetate and the pooled organic phases were washed with water(30 ml) containing sodium chloride (10 g).

The organic phases were then anhydrified on magnesium sulphate anddried; a 8.9 g residue was obtained (yellow oil) which solidifies forcooling at 0° C. By crystallization from isopropilic ether 6.5 g ofANMPE were obtained at the pure state. The ¹ H NMR (CDCl₃) (80 MHz)analysis gave the following data: 2.34 (3H, s); 5.45 (2H, s); 7.05-7.75(7H, m); 8.24 (1H, dd).

Example 4 Pharmacological Examples

The products prepared above were characterized by a pharmacologicalviewpoint.

In the in vivo studies (for example toxicity) the products obtainedabove were administered in form of suspension in carboxymethylcellulose1-2% by weight.

For the in vitro tests (piastrinic tests) the nitroderivatives, 1 mmol,were dissolved in dimethylsulfoxide and then diluted according to theconcentrations listed in the Table.

Aspirin ASA 30 mmols was dissolved in a mixture of etanol:H₂ O in therange 1:10 by volume and then diluted according to the concentrationslisted in the Table.

The samples, obtained without adding the substance under examination(ASA, ANBE, ANMPE), did not show any significant reply.

Toxicity

The acute toxicity was evaluated through oral subministration of asingle dose of 1, 3, 10, 30, 100, 200 mg/Kg of product in groups of 10little rats.

The lethality incidence and the appearance of toxic sinthomatology werenoted within a period of 14 days. Also after administration of a dose of200 mg/Kg the animals did not show any apparent toxicity both with ANMPAand with ANBE.

Tolerability

The gastric tolerability was evaluated through oral subministration inthe rat measuring the seriousness of the gastropathie induced accordingto the criterium indicated by Wallace et al. (Am. J. Physiol. 259, G642,1990).

Piastrinic Tests

Anti-Aggregating Piastrinic Activity (Anti-thrombotic Activity)

The anti-aggregating piastrinic activity was evaluated in vitro on humanpiastrines stimulated by trombin or by arachidonic acid according to themethod described by Bertele et al. (Science 220, 517, 1983).

COX Inhibition (Anti-inflammatory Activity)

The inhibition activity of the cyclooxygenases was determined in humanpiastrines according to the method described by Patrono et al(Thrombosis Res. 17, 317, 1980). The enzimatic activity was expressed aslevel of Tromboxan B2 (T_(X) B2) and measured in ng/ml.

Piastrinic Adhesion

The inhibition activity of the piastrinic adhesion was evaluatedaccording to the method described by Bellavite et al. (Anal. Biochem.216, 444, 1994).

Intracellular Piastrinic Calcium

The effect of the compounds of the invention or comparison compounds onthe calcium concentration inside the piastrine was measured according tothe method of Pollock et al. (Biochem. J. 235, 869, 1986).

                                      TABLE 1                                     __________________________________________________________________________                       PIASTRINIC AGGREGATION (%).sup.(1)                                            INDUCED BY INDUCED BY                                      COMPOUND                                                                              CONCENTRATION(M)                                                                         ARACHIDONIC ACID                                                                         TROMBIN                                                                              COX INHIBITION (%).sup.(1)               __________________________________________________________________________    ASA (Ex. 1)                                                                           10.sup.-5  40         100    --                                       ASA (Ex. 1)                                                                           5.10.sup.-5                                                                              --         --     3                                        ASA (Ex. 1)                                                                           10.sup.-4  0          100    1                                        ASA (Ex. 1)                                                                           10.sup.-3  --         80     --                                       ANBE (Ex. 2)                                                                          10.sup.-5  100        --     80                                       ANBE (Ex. 2)                                                                          5.10.sup.-5                                                                              --         --     70                                       ANBE (Ex. 2)                                                                          10.sup.-4  50         60     30                                       ANBE (Ex. 2)                                                                          10.sup.-3  20         50     --                                       ANMPE (Ex. 3)                                                                         10.sup.-5  60         70     5                                        ANMPE (Ex. 3)                                                                         5.10.sup.-5                                                                              10         40     2                                        ANMPE (Ex. 3)                                                                         10.sup.-4  0          0      --                                       __________________________________________________________________________     .sup.(1) % REFERRED TO CONTROLS (FOR ADDING OF THE AGGREGATING SUBSTANCE      ONLY)                                                                    

RESULTS

From the results of the Table, it can be seen that ANMPE (compound ofthe invention) is much more efficient with respect to ASA and ANBE inthe inhibition of the piastrinic aggregation induced by arachidonicacid. In the case of ANMPE it is higher than ANBE and similar to ASA.

Nevertheless in the piastrinic aggregation induced by trombin, whichhigher patogenetic value is known with respect to the arachidonic acidor other aggregating stimulus, the ANMPE gives values surprisinglyhigher both with respect to ANBE and ASA.

For the COX inhibition properties, the product of the invention ANMPEshows activities similar to ASA, but well higher with respect to ANBE.

This is much more surprising if we consider that ANMPE as well as ANBE,but differently from ASA, it is very well tolerated in the gastricmucose.

Indeed the gastric tolerance tests have shown that already at doses of50-100 mg/Kg ASA induced severe damages in the intestinal gastric mucoseof the treated animals. On the contrary ANMPE and ANBE, also whenadministered at doses of 250-500 mg/Kg did not produce relevant damages.

As regards the other piastrinic tests:

piastrinic adhesion and intracellular piastrinic calcium, only ANMPEresulted efficient in inhibiting significantly and in a dose-dependentway (from 10⁻⁵ to 10⁻⁴ M) both pathological processes.

On the contrary it was not possible to see any inhibiting effect withother compounds under examination.

We claim:
 1. A compound of the formula:

    A--X.sub.1 --NO.sub.2

or salts, wherein; A=R(COX)_(t), wherein t=0 or 1; X=O, NH, NR_(1c),wherein R_(1c) is a linear or branched C₁ -C₁₀ alkyl group ##STR11##wherein m and n are an integer from 1 to 6; wherein R_(2a) is H or CH₃ ;and wherein the linking with X₁ can be in any position of the ring; Ris: ##STR12## wherein: X₀ =X;R₁ is OCOR₃ ; wherein R₃ is methyl, ethyl,linear or branched C₃ -C₅ alkyl, or the residue of an etherocycle withonly one ring having 5 or 6 atoms which can be aromatic, partially orcompletely hydrogenated, containing one or more ethero-atoms selectedindependently among O, N and S; R₂ is hydrogen, hydroxy, halogen, linearor branched C₁₋₄ alkyl, linear or branched C₁ -C₄ alkoxyl; a linear orbranched C₁ -C₄, perflouroalkyl, nitro, amino, mono- or dialkylaminewith C₁₋₄ alkyl; or R₁ and R₂ together are dioxymethylene group; n_(I)is an integer 0 or 1; X₁ in the formula A--X₁ --NO₂, is a bivalentconnecting bridge selected from the group consisting of --YO-- where Yis a linear or branched C₁₋₂₀ alkylene, or a substituted orunsubstituted C₅₋₇ cycloalkylene; and ##STR13## wherein n is an integerfrom 1 to 6, R_(2a) is an defined above; Y is as defined above, and r=0or 1; with the proviso that when X=NH, then Y is only ethylene and R₂ ishydrogen; and with the proviso that R₁ cannot be OCOR₃, in position 2when R₃ is methyl.
 2. The compound according to claim 1, wherein R is##STR14## wherein --X₁ --NO₂ is --CH₂ --ONO₂, --CH₂ CH₂ CH₂ CH₂ ONO₂,--CH₂ CH₂ ONO₂, --CH₂ CH₂ OCH₂ CH₂ ONO₂, or --CH₂ CH₂ OCH₂ ONO₂.
 3. Thecompound according to claim 1, wherein X=NR_(1c) wherein R_(1c) is alinear or branched C₁ -C₄ alkyl group.
 4. A composition comprising thecompound according to claim 1 or claim 2 and a pharmaceuticallyacceptable excipient.
 5. A method for the treatment of inflammationcomprising administering the composition of claim
 4. 6. A method for thetreatment of thrombosis comprising administering the composition ofclaim
 4. 7. A method for the treatment of septic shock comprisingadministering the composition of claim 4.